Advanced Microbial Physiology

1. Advanced Microbial Physiology Lecture 1 Minimum Bacterial Genome

2. Definitions: • Genome – the sum of total genes within a species of an organism • Essential genes – genes absolutely required for growth and survival • Non essential genes – genes whose destruction does not lead to significant growth defects in a cell.

3. Why study essential genes? • Essential genes are important for cellular function and physiology; to study them will reveal details about microbial physiology • Practical application: essential genes encode essential proteins which are excellent drug targets to develop new antibiotics

4. Strategies for Essential ID • Saturated transposon mutagenesis • Antisense expression controlling gene expression • Systematic gene knock-out (or inability to knock-out)

5. Nonhomologous recombination • No requirement for two DNAs being of the same or similar nucleotide sequences • Needs enzymes that recognize specific regions in DNA • Mechanisms include: • transposition • phage integration and excision • resolution of cointegrates

6. Transposition • Transposons – DNA elements that can hop (transpose) from one place in DNA to another • Transposons are known to exist in all organisms on earth • Movement by a transposon is called transposition, catalyzed by enzymes called transposases • Transposons usually encode their own transposases

7. Transposition • Many transposons are essentially cut out of one DNA and inserted into another • Other transposons are copied and then inserted elsewhere • Donor DNA and target DNA

9. Structure of a Bacterial transposon

10. Structure of Bacterial Transposons • All contain repeats at their ends, usually inverted repeats (IR) • Presence of short direct repeats in the target DNA that bracket the transposon • The sites of insertion are different among target DNAs

11. Types of Bacterial Transposons • Smallest bacterial transposons are called insertion sequence elements (IS elements); they only encode transposase enzymes • Composite transposons – formed by two IS elements of the same type, bracketing other genes

12. Composite transposons

19. Antisense expression • Antisense RNA expression. Random cloning and expression of short pieces of genomic DNA on a plasmid in an microorganism to elucidate the function of the genes

20. Plasmid DNA Protein Antisense RNA mRNA mRNA DNA DNA Antisense cell Normal cell X Conditional Antisense Inhibitionof Protein Synthesis Inducible promoter No protein

21. Millions of random DNA fragments Essential gene blocked by antisense Non essential gene blocked by antisense No cell growth Essential Protein mRNA DNA Shotgun Antisense Expression Determines Essentiality of Genes Pathogen genome

22. Ultra-Rapid Functional Genomics Identify >100 essential gene drug targets per month Antisense (+ inducer) No antisense (- inducer)

24. Gene Knock-out • Gene replacement (knock-out): also known as reverse genetics. The purpose is to remove (knock-out) most of one gene and see what happens to the phenotype of the organism. Suicide vector is used.

30. Number of essential genes determined for various bacterial species.